DESCRIPTION (Verbatim from the application): The cardioprotective effect of estrogen is complex, and incompletely understood. We have identified one potentially important action of physiological circulating estrogen on coronary arteries, which involved an elevation of basal nitric oxide release from the endothelium and an ensuing smooth muscle relaxation through activation of one type of calcium-sensitive (large conductance; BK) potassium channel in the smooth muscle. Our results indicate that estrogen causes fundamental alterations in Ca2+ signaling in the coronary endothelium, which leads to an elevation of nitric oxide production, which in turns alters Ca2+ signaling in the smooth muscle. This proposal focuses on novel, interlinked Ca2+ signaling mechanisms to explain the effects of estrogen and nitric oxide on coronary artery diameter. Specifically, Aim 1 will determine the mechanisms by which estrogen leads to an elevation of endothelial [Ca2+]i; Specific Aim 2 will explore positive feedback regulation of endothelial Ca2+ by local and global Ca2+ signaling from ryanodine receptors (RyRs) in the endoplasmic reticulum to small conductance calcium-sensitive (SK) and BK channels; Specific Aim 3 will determine the negative feedback mechanisms by which endogenously produced nitric oxide activates RyRs and BK channels in coronary artery smooth muscle, with a focus on the key roles of phospholamban, which regulates SR Ca2+-ATPase activity, and on the beta-subunit of the BK channel. To address these issues, we have developed techniques to measure global and local calcium in the endothelium and smooth muscle of intact pressurized coronary arteries, including coronary arteries from phospholamban and beta-subunit gene-ablated mice, and developed novel, selective peptide inhibitors of cGMP-dependent protein kinase. The proposed study should significantly deepen our understanding of the regulation of Ca2+ signaling in coronary arteries, and the important influences of physiological estrogen and nitric oxide on coronary artery function. This work should also suggest novel mechanisms for therapeutic interventions to mimic the beneficial effects of estrogen and nitric oxide on calcium signaling in coronary arteries.